Brain MRI segmentation of Zika-Exposed normocephalic infants shows smaller amygdala volumes

Background Infants with congenital Zika syndrome (CZS) are known to exhibit characteristic brain abnormalities. However, the brain anatomy of Zika virus (ZIKV)-exposed infants, born to ZIKV-positive pregnant mothers, who have normal-appearing head characteristics at birth, has not been evaluated in detail. The aim of this prospective study is, therefore, to compare the cortical and subcortical brain structural volume measures of ZIKV-exposed normocephalic infants to age-matched healthy controls. Methods and findings We acquired T2-MRI of the whole brain of 18 ZIKV-exposed infants and 8 normal controls on a 3T MRI scanner. The MR images were auto-segmented into eight tissue types and anatomical regions including the white matter, cortical grey matter, deep nuclear grey matter, corticospinal fluid, amygdala, hippocampus, cerebellum, and brainstem. We determined the volumes of these regions and calculated the total intracranial volume (TICV) and head circumference (HC). We compared these measurements between the two groups, controlling for infant age at scan, by first comparing results for all subjects in each group and secondly performing a subgroup analysis for subjects below 8 weeks of postnatal age at scan. ZIKV-exposed infants demonstrated a significant decrease in amygdala volume compared to the control group in both the group and subgroup comparisons (p<0.05, corrected for multiple comparisons using FDR). No significant volume differences were observed in TICV, HC, or any specific brain tissue structures or regions. Study limitations include small sample size, which was due to abrupt cessation of extramural funding as the ZIKV epidemic waned. Conclusion ZIKV-exposed infants exhibited smaller volumes in the amygdala, a brain region primarily involved in emotional and behavioral processing. This brain MRI finding may lead to poorer behavioral outcomes and warrants long-term monitoring of pediatric cases of infants with gestational exposure to Zika virus as well as other neurotropic viruses.


Methods and findings
We acquired T2-MRI of the whole brain of 18 ZIKV-exposed infants and 8 normal controls on a 3T MRI scanner. The MR images were auto-segmented into eight tissue types and anatomical regions including the white matter, cortical grey matter, deep nuclear grey matter, corticospinal fluid, amygdala, hippocampus, cerebellum, and brainstem. We determined the volumes of these regions and calculated the total intracranial volume (TICV) and head circumference (HC). We compared these measurements between the two groups, controlling for infant age at scan, by first comparing results for all subjects in each group and secondly performing a subgroup analysis for subjects below 8 weeks of postnatal age at scan. ZIKV-exposed infants demonstrated a significant decrease in amygdala volume compared to the control group in both the group and subgroup comparisons (p<0.05, corrected for multiple comparisons using FDR). No significant volume differences were observed in TICV, HC, or any specific brain tissue structures or regions. Study limitations include small sample size, which was due to abrupt cessation of extramural funding as the ZIKV epidemic waned. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Introduction
Although the Zika virus (ZIKV) epidemic has subsided markedly, the impact of congenital ZIKV infection on the developing brain of children who were prenatally infected is still not completely understood. In infants with congenital Zika syndrome (CZS), microcephaly and other macro-anatomical brain abnormalities have been confirmed using ultrasound, CT, and conventional MR imaging [1][2][3][4][5][6][7][8]. However, a larger proportion of infants who were prenatally exposed to ZIKV (ZIKV-exposed), due to maternal ZIKV infection during pregnancy, are normocephalic at birth without apparent clinical or macro-anatomical brain abnormalities [9]. Emerging reports suggest that these ZIKV-exposed infants may develop diminished head circumference months after birth [10]. A recently published report indicates that 32% of ZIKVexposed children, up to age 3, have below-average neurological development [11]. Extensive brain malformations and neurological abnormalities are seen in these children, which are thought to be secondary to microstructural damage caused by ZIKV on the developing fetal brain [11].
The aims of this prospective study were (a) to examine the radiological manifestations in infants exposed to maternal ZIKV infection who demonstrate no microcephaly or other gross macroanatomical abnormalities, (b) to quantify specific brain tissue type volumes in ZIKVexposed and matched control infants, and (c) to make between-group comparisons. Analysis of the brain imaging measures will provide a baseline for longitudinal neuroimaging assessments in this population.

Subjects
This prospective study is HIPAA-compliant and was approved by the Institutional Review Board of the University of Miami Miller School of Medicine and the Jackson Health System, Miami, Florida. Both ZIKV-exposed and healthy control infants were recruited from the Jackson Memorial/Women's Hospital and Holtz Children's Hospital services, Miami, Florida. Parents of all infants included in this study provided written informed consent.
For the purposes of this study, ZIKV exposure was defined as a positive ZIKV specific immunoglobin M (IgM) enzyme-linked immunosorbent assay (ELISA) result in maternal serum without microcephaly in the infant. Conventional plaque reduction neutralization testing (PRNT) was performed for the detection of Zika virus and Dengue virus. Infants with ZIKV infection were defined as infants with characteristic findings associated with CZS including microcephaly, gross ventriculomegaly, and parenchymal calcifications; such infants were excluded from this study. Microcephaly was defined as head circumference more than 2 standard deviations below the mean for gestational age and sex [12]. The inclusion criteria for the ZIKV-exposed infant group included mothers with confirmatory Zika testing by the Florida Department of Health (i.e., IgM-positive and confirmatory PRNT results). Infants whose mothers refused testing for Zika were excluded from the study. Infants whose mothers had other evidence of viral or parasitic infection such as HIV, syphilis, rubella, cytomegalovirus, and toxoplasmosis during pregnancy were also excluded.
A total of 49 infants were recruited for the study and 36 completed the MRI scans. Of the remaining subjects, 10 were excluded for being above 20 weeks postnatal age at scan to focus on young infants. The final study cohort for this report consists of 18 ZIKV-exposed and 8 healthy control infants. Mothers of the participating infants were screened at enrollment for major depression during pregnancy using the Edinburgh Postnatal Depression Scale (EPDS) and for use of anti-depressive medication.

MR imaging and volumetric measurements
Infants were scanned without sedation and during natural sleep time on a 3T MRI scanner (General Electric) using a 32-channel head coil. They were restrained on a papoose board and monitored with EKG and pulse oximeter during the scan. High-resolution axial T2-weighted MR images of the brain were obtained using a T2-PROPELLER sequence (TE: 68.7 ms; TR: 13.6 s; slice thickness: 2 mm; in-plane spatial resolution: 0.625 x 0.625 mm 2 ; voxel volume: 0.625 x 0.625 x 2 = 0.78 mm 3 , acquisition time = 2 minutes 15 seconds). The use of this sequence has helped to reduce the effect of subject motion and magnetic susceptibility artifacts in images as the infants were scanned without sedation. The protocol also included axial susceptibility weighted imaging, proton MR spectroscopy, diffusion-weighted imaging, T1-weighted FLAIR, and T1-weighted BRAVO sequences for a total acquisition time of approximately 25 minutes.
Brain images were processed using the Morphologically Adaptive Neonate Tissue Segmentation (MANTiS) program [13]. MANTiS is a tissue segmentation toolbox developed for the Statistical Parametric Mapping (SPM) software. It segments T2-weighted brain images of infants using adaptive templates into 8 different tissue types defined as distinct anatomical regions of interest (ROI) and include white matter (WM), cortical grey matter (GM), deep nuclear GM, corticospinal fluid (CSF), amygdala, hippocampus, brainstem, and cerebellum (Fig 1). Radiologists with at least three years of experience reviewed all images for quality and assessed these diagnostically. The radiologist co-authors reviewed the ROIs segmented by the MANTiS for accuracy of the tissue types (i.e., grey matter or white matter) or anatomical delineations of each ROI and manually edited segmented ROIs as needed. The most performed manual edits were for removal of erroneously segmented extracranial soft tissues and incomplete segmentation of the cerebellum. We noted incomplete imaging of the posterior fossa in two subjects that were excluded from the calculation of cerebellum and brainstem volumes. We calculated the volumes of all eight regions from the segmentation results by multiplying each ROI's voxel count by the voxel size. For each subject, we calculated the total intracranial volume (TICV) by adding the volumes of all eight segmented ROIs. In order to reduce the effects of variability due to individual head size differences, we normalized each subject's ROI volumes by dividing them by TICV. As such, TICV is reported in absolute volume (cm 3 ), while the other measurements are reported as normalized volumes as a percentage of TICV between 0 and 1.

Statistical analysis
Between-group differences in demographics and EPDS scores were checked using Wilcoxon and Chi-squared tests for continuous and categorical variables, respectively. If any of the gestational age, age at scan, or postmenstrual age were found to be significantly different, these variables were controlled for as covariates in the analysis of head circumference and the 9 brain volumetric measurements (8 ROIs + TICV). Measurement outcomes were first checked for normality of residuals with Shapiro-Wilk tests, and for homogeneity of residual variance with Levene's tests. We also tested for homogeneity of regression slopes to determine if there were significant interactions between the covariate and the grouping variable. If those conditions were met, between-group comparisons were performed using analysis of covariance (ANCOVA) at 95% confidence level (p < 0.05) corrected for multiple comparisons using false-discovery rate (FDR) [14]. Otherwise, if assumptions of normality and homogeneity were not met, non-parametric ANCOVA procedures were used instead. Finally, a correlation analysis was performed between the neuroimaging measurements and EPDS scores to find associations between the depression level of mothers and the brain developmental outcomes of their infants. All statistical tests were performed using the R programming language.

Results
Demographic information for the ZIKV-exposed and healthy control groups are provided in Table 1A. Infants from both groups were matched for gestational age (GA), with full-term pregnancies for all controls, with only 3 ZIKV-exposed infants born preterm at 35 and 36 weeks GA and were not significantly premature to exclude from the study. The two groups did differ in their age at scan with slightly older ZIKV-exposed babies. This, however, did not impact the overall postmenstrual age (PA) which was not significantly different. Both groups were also matched for term gestation, sex, and race with subjects primarily from black or white Hispanics ethnic backgrounds. Mothers of ZIKV-Exposed infants did not exhibit signs of higher depression during pregnancy than mothers of controls, with only one mother in each group showing major depression (EPDS > 13) and only one mother of a ZIKV-Exposed infant was taking anti-depressive medication.
Since age at scan was the only variable that differed between ZIKV-exposed and control infants, we controlled for age at scan as a covariate in the subsequent analysis of head circumference and brain volumetric measures. We also performed a subgroup analysis by selecting subjects below 8 weeks of age at scan from both groups to obtain an age-matched sample. This resulted in 9 ZIKV-exposed and 7 controls who showed no significant differences in any of the demographic characteristics (Table 1B).
ANCOVA analysis showed no significant interactions (p > 0.05) between age at scan and the grouping variable (Fig 2), confirming that the homogeneity of slopes assumption was met, and the difference in age did not influence the outcome in measurements between controls and ZIKV-exposed subjects. We observed significant decreases in volume in the ZIKVexposed group only in the amygdala (controls: 0.0036; ZIKV-exposed: 0.0027; p = 0.013). There were no appreciable differences in the overall size of the brain measured by TICV and HC, or in any other brain structures (Fig 3). The subgroup analysis showed identical results, Table 1

PLOS ONE
Zika-Exposed infants show smaller amygdala volumes with homogeneity of slopes (p > 0.05) when controlling for age (Fig 2), and between-group differences only observed in the amygdala (controls: 0.0038; ZIKV: 0.0026; p = 0.003) while other measures did not significantly differ. Results of the correlations between the EPDS scores and TICV, HC, and the remaining volumetric measures did not show significant associations between depression experienced by the mothers and the brain development of their infants.

Discussion
To the best of our knowledge, this is the first study to segment brain T2-MRIs obtained from ZIKV-exposed infants into anatomical regions, component tissue types, and CSF. The main finding from this prospective case-control study is that there was a significant volume reduction in the amygdala for the ZIKV-exposed group when evaluating both the complete ZIKVexposed cohort and its subgroup below 8 weeks of age. There is no published report in the literature on decreased amygdala volume or its underlying pathological causes in ZIKV-exposed human infants. We therefore looked for findings from animal model ZIKV infection studies [15,16] as well as human studies [17][18][19][20][21][22] on associations between psychological distress or depression experienced by mothers during the pregnancy and the amygdala volume in infants born to them for corroboratively interpreting the results of this study. Longitudinal studies of postnatally ZIKV-infected infant macaques at 3, 6, and 12 months of age have shown smaller amygdala volumes and reduced functional connectivity in amygdala-connected brain networks, which regulate emotional behavior and functions [15,16]. Furthermore, these changes to the amygdala in ZIKV-infected macaques corresponded with changes in their emotional responses to stress inducing stimuli [15,16]. However, findings in the literature on associations between the amygdala volume of human infants and psychological stress experienced by their mothers during pregnancy were inconsistent [17][18][19][20][21][22]. Reports show lower amygdala volumes in male neonates [17] and 4 year old children [18,19], but larger amygdala volumes in girls [19,20], associated with higher prenatal maternal distress, depressive symptoms, and anxiety during pregnancy. Larger amygdala volumes and lower functional connectivity were also found in newborn infants of mothers who experienced prematernal distress due to Covid-19 infection [21], while investigation of the amygdala's microstructure in neonates showed no relation to the mother's anxiety [22]. Our results showed that mothers of ZIKV-exposed babies did not experience higher levels of depression during pregnancy, with only one mother in each group exhibiting major depression (EPDS > 13), and that higher EPDS scores did not correlate with smaller amygdala volumes or other measurements. Since higher-level cognitive and functional deviations are often difficult to assess in infants, it is possible that specific disabilities will become more evident with increasing age. Several two to three-year follow-up studies of normocephalic ZIKV exposed infants, including a metaanalysis review that used data from 22 published articles, have indicated neurodevelopmental delays in language, cognition, and motor skills [23][24][25], deficits in the visual system [26], and low growth velocity with altered neurological and psychomotor development [27]. However, a small number of studies have also reported no risk of neurodevelopmental delays [28,29]. Therefore, while most of the brain regions examined in our study do not appear to be abnormal in size, it is possible that structural abnormalities in ZIKV-exposed infants who were normocephalic at birth will become more evident at a later stage when neurodevelopmental delays will become more apparent. There is, in fact, a paucity of published neuroimaging studies with large sample sizes evaluating brain-imaging outcomes in this population beyond 3 years of age. A recent paper, which aggregated over 100,000 MRI scans to map growth trajectories of brain tissue types (i.e., cortical GM, WM, and deep GM) across the human lifespan, revealed that cortical GM growth peaked at nearly 6 years of age, with variability in cortical GM peaking at 4 years of age, while subcortical GM and WM peaked at 14.4 and 28.7 years of age, respectively [30]. These growth peaks in cortical GM were observed to be 2 to 3 years later than previously reported, which suggests that neuroanatomical developmental deficits in ZIKV-exposed infants may not manifest until at least 6 years of age. Furthermore, ZIKV exposed normocephalic newborns were found to have higher plasma concentrations of lysophosphatidylcholine relative to controls [31], which could contribute to a wide spectrum of clinical phenotypes such as neurodevelopmental, neurocognitive and ophthalmological abnormalities in childhood beyond microcephaly. Therefore, it is imperative to perform longitudinal neuroimaging and neurodevelopmental follow-ups of ZIKV-exposed infants to validate these early findings, but also to reassess the long-term clinical impact of ZIKV.
None of the remaining measurements we collected showed statistically significant betweengroup differences, although ZIKV-exposed infants appear to have higher CSF and WM volumes and concomitantly lower cortical and deep GM volumes. However, the significance of these observations is somewhat undermined by the sample size and the observed relatively higher standard deviations in the ZIKV-exposed group that reduced the statistical power of our study.
There are a few limitations to this study. As previously mentioned, first is the small sample size of ZIKV-exposed (n = 18) and control (n = 8) subjects. We were unable to enroll additional participants due to funding constraints. The waning ZIKV epidemic in our South Florida region and the nation led to an abrupt cessation of funding for this study by the Florida State Department of Health. Additionally, optimal assays and specimens used for testing, and the timing of testing for congenital ZIKV infection were unknown. The PRNT cannot distinguish between maternal and infant antibodies in specimens collected from infants at or near birth, and thus it was not performed on infants included in this study. Based on what is known about other congenital infections, maternal antibodies are expected to become undetectable by 18 months of age and might become undetectable earlier [32].
In summary, our study indicates that while overall brain volume measurements from MRIs performed on otherwise normal-appearing ZIKV-exposed infants indicate no significant change, a significant decrease in amygdala volume was still observed. Future longitudinal studies with a larger number of subjects and correlations with neurodevelopmental test scores would be needed to better understand the clinical and behavioral impact of ZIKV infection on pregnant mothers and ZIKV-exposure to their babies. If our results are reproduced in a larger study, then diagnostic evaluation and clinical management of these infants may warrant development of effective interventions.
Supporting information S1 Table. Original data including age, EPDS scores, and brain measurements across all study participants. (XLSX)